Interstitial cells of Cajal (ICCs), pacemaker and neuromodulator cells in the gastrointestinal (GI) tract, play an important role in GI motility. However, quantifying ICCs is challenging due to their mixed morphologies. Here, we present a protocol for preparing and immunostaining ICC in the murine gastric tunica muscularis using artificial intelligence (AI). We describe steps for obtaining muscles, whole-mount staining, and imaging ICC using confocal microscope. We then detail procedures for training an AI to identify ICCs and quantify their volume. For complete details on the use and execution of this protocol, please refer to Taheri et al.